Module Overview. Lecture. DNA library synthesis (PCR) Introduction

Transcription

1 Module Overview Day 1 Lecture Introduction Lab DNA library synthesis (PCR) SELEX I: Building a Library SELEX II: Selecting RNA with target functionality SELEX III: Library deconvolution, problem-solving & technical advances Characterizing aptamers Introduction to porphyrins: chemistry & biology Aptamer applications in biology & technology Aptamers as therapeutics DNA library purification (agarose gel electrophoresis) RNA library synthesis (In vitro transcription = IVT) RNA purification and heme affinity selection RNA to DNA by RT-PCR Post-selection IVT Journal Club 1 Aptamer binding assay Journal Club 2

2 SELEX III Lecture 4 15 February, 2011

3 Summary Developed a conceptual framework for SELEX Library diversity Calculations Maximizing diversity within technical constraints Choosing the appropriate library for your needs! Examined some key steps involved in the process: Target selection RNA library construction Partitioning strategies SELEX can be successfully executed on: Very distinct targets Using distinct library design (diversity, representation, etc) Using distinct partitioning strategies Fairly robust and generally applicable strategy

4 Today s Objectives Deconvoluting a SELEX library How do you know you ve succeeded (or failed)? Conceptualizing selection stringency Things to consider if/when SELEX fails

5 A typical SELEX workflow unique molecules RNA Library Selection column Immobilized target In vitro transcription Wash to eliminate non-binders Elute survivors Aptamer enriched RNA library RT-PCR DNA library Deconvolution of the selected library

6 Deconvoluting your selected library Was your SELEX experiment successful? Have you obtained your desired aptamers How do you determine this? If your SELEX was successful: How do you identify the individual members of the selected library? Are all members of your library competent for target binding? Are there discernible, conserved features present in your aptamers?

7 Determining the success of your SELEX experiment Compare library dissociation constants pre- and post- SELEX What does it mean to have a larger dissociation constant or K d? Schneider et al, FASEB J,, 7(1), , 1993

8 Determining the success of your SELEX experiment Track the amount of RNA recovered at the end of each round of selection Advantages: Determine progress in real time Facilitates rapidly knowing the impact of changing a variable during SELEX Disadvantage Introduce radioactivity in your workflow

9 Library deconvolution Achieve: Isolation of individual aptamers to simultaneously facilitate: -> Sequencing (identification) -> Characterization (binding, etc) Aptamer 1 Aptamer 2 Aptamer mixture 1,2,3,4 Aptamer 3 Aptamer 4

10 Library deconvolution You observe binding of your bulk selected library to the target ~ unique members in starting library How many present at the end? Identifying individual aptamers in your library How would you do this? Exactly how you d clone a new gene!

11 Cloning the aptamer library Restriction site 1 Restriction site 2 T7 promoter Fixed Region 1 Fixed Region 2 Single hit conditions: One insert on average incorporated into one plasmid Each plasmid now encodes a single aptamer Problem You have a mixture of plasmids How do you isolate clonal plasmids? Addison Wesley Longman, Inc.

12 Cloning the aptamer library Bacterial transformation Single hit conditions: Transform into Bacteria On average: 1 plasmid per bacterial cell Plate on selective solid media Plating on selective media: Single colony derived from a single bacterial cell LB Agar + antibiotic plate Each colony contains many bacterial cells, each carrying the identical plasmid

13 Aptamer library now encoded in plasmid library Glycerol stocks -80ºC) Achieved: Mixture of aptamers in selected library resolved into a plasmid library of individual aptamers Preserved ability to manipulate library Library archive Mini-prep to isolate plasmid Aptamer sequencing In vitro transcription to obtain aptamer

14 but what went wrong with my SELEX? some common scenarios 1. No detectable binding to target Why might this occur? Problem with your binding assay How might you assess this? Too few rounds of selection completed How would you determine this? Your selection process went awry Poor choice of selection stringency conditions Sequences selected based on amplification efficiency, NOT target binding PCR, RT, in vitro transcription

15 but what went wrong with my SELEX? Some common scenarios 2. Selected library and individual aptamers bind tightly to target, but ONLY when immobilized in the format used during SELEX Why might this arise? Aptamers partially or completely recognize and bind to the solid support! How would you change your selection format to counter this?

16 Eliminating library members with high inherent affinity for solid support unique molecules RNA Library unique molecules RNA Library Selection column Immobilized target In vitro transcription Negative selection column Solid support only Wash to eliminate non-binders Elute survivors Positive selection column Immobilized target Aptamer enriched RNA library RT-PCR DNA library Solution: Introduce a negative selection step

17 Maximizing SELEX efficiency Desirable: Obtain target aptamers on first try! In the fewest possible number of rounds What is the best way to ensure achieving this? Efficiently eliminate non-binders Efficiently recover binders Driven by selection stringency!

18 Conceptualizing stringency during SELEX Molecular targets e.g. heme Majors SELEX Strategy for efficiently querying your RNA library Barton Strategy for efficiently querying the MIT Collections History Science Engineering Philosophy Book collection = RNA sequence collection Sub-topic Book {x,y,z} = Aptamer Book X Book Y Book Z

19 Conceptualizing stringency during SELEX Trying to locate that {Thermodynamics textbook} used in {20.110} Limited specific information available Perform a low stringency search

20 Conceptualizing stringency during SELEX Trying to locate that {Thermodynamics textbook} used in {20.110} Limited specific information available Narrow using available information Use too narrowly defined a search term Result: Lose your desired target!

21 Conceptualizing stringency during SELEX Trying to locate that {Thermodynamics textbook} authored by {Dill} used in {20.110} Narrow using available information

22 Conceptualizing stringency during SELEX Trying to locate that {Thermodynamics textbook} authored by {Dill} used in {20.110} Narrow using available information More specific information about target available Result: More efficient search and recovery!

23 Conceptualizing stringency during SELEX MIT Libraries Trying to locate that {Thermodynamics textbook} used in {20.110} RNA Library Trying to find the {RNA aptamers} that bind {target X} The more information initially specified, the more efficient the search for aptamers (see next slide) Very little information specified in initial query Difficult to rationally restrict the search space Searching is inherently inefficient How can we modulate information input to influence the outcome of our SELEX experiment?

24 SELEX à la Tuerk & Gold Target T4 DNA polymerase Structure for residues from the PDB ( Target known to interact with RNA from prior work Sequence below found in the mrna encoding the T4 DNA polymerase Regulatory mechanism: T4 DNA polymerase binds its own mrna decreases its own synthesis 8 nucleotides [AAUAACUC] are critical for the interaction What underlies the preference for this loop sequence? Based on objective, what library design would you choose? C. Tuerk and L. Gold; Science; 249 (4968), , 1990

25 Modulating SELEX stringency--practically 1. Vary how extensively the selection column is washed to remove non-interacting RNAs Higher stringency --> more washes Lower stringency --> fewer washes Information content specified: Thermodynamics (Dissociation constant) The lifetime of the {aptamer-target} complex must exceed the time it takes to complete your washing Sufficient complex must survive the dilution and extraction process associated with washing Query: Find the {RNA aptamers} that bind {target X} with a {dissociation constant xx}.

26 Modulating SELEX stringency--practically 2. Alter the [library]:[target] ratio Higher stringency --> higher ratio Lower stringency --> lower ratio Information content specified: Thermodynamics (Dissociation constant) Limit the number of possible target binding sites Favor recovering higher affinity library members (increased signal) Fewer sites for non-specific and low affinity interactions (decreased noise) E.g. Less solid support used when the amount of target used is decreased Query: Find the {RNA aptamers} that bind {target X} with a {dissociation constant xx}.

27 Modulating SELEX stringency--practically 3. Using buffer additives to suppress undesired interactions ph Consider target pi ph too low --> target carried net positive charge --> encourage nonspecific electrostatic interactions with negatively charged RNA Raising ph increases stringency by reducing net positive charge on target since this reduces bulk library interactions with the target trna Bind non-specific sites on solid support Salt concentration Modulate electrostatic contributions during binding Major benefit is in reducing the noise during your selection

28 My parameter optimization space is HUGE help!? Vary: Wash number [Library]:[target] ratio Buffer conditions ph [salt] trna BSA (protein) Where do I start my SELEX? Which variable(s) do I change if it fails?

29 Library synthesis (DNA synthesizer) Enzymatic reactions PCR (thermal cycler) RT (thermal cycler) In vitro transcription (thermal cycler) Binding reactions 96-well plates (shakers) Automating SELEX Inter-process sample transfer Liquid handling robots Cox & Ellington, Bioorganic & Medicinal Chemistry, 9(10), , 2001

30 Summary Selected aptamer libraries can be made into plasmid libraries Using standard molecular biology methods Each plasmid represents a specific aptamer in selected pool Facilitate aptamer archival and further characterization Many factors can impact the success or failure of SELEX Must carefully consider target properties in selecting your SELEX conditions Establish your strategy for using stringency to control the efficiency of your selection Selecting a stringency protocol is empirical Insufficient initial knowledge to rationally decide best strategy beforehand Altering stringency involves considering thermodyamic principles